A Novel Role for Protein Phosphatase 2A in Receptor-mediated Regulation of the Cardiac Sarcolemmal Na+/H+ Exchanger NHE1

Abstract

G q protein-coupled receptor stimulation increases sarcolemmal Na ؉ /H؉ exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A 1 receptor stimulation inhibits this response through a G i proteinmediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A 1 receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the ␣ 1 -adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A c ) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G i proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A c and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A c dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A 1 receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G i protein-mediated translocation of PP2A c and NHE1 dephosphorylation.The cardiac sarcolemmal Na ϩ /H ϩ exchanger (NHE) 2 is a membrane glycoprotein encoded by the NHE1/SLC9A1 gene (1), which is one of nine known members of the solute carrier 9 (SLC9) gene family (2). The sarcolemmal NHE contributes significantly to the control of intracellular pH (pH i ) in cardiac myocytes, particularly in response to intracellular acidosis (3). Although basal activity of the sarcolemmal NHE is low under physiological conditions (3), increased exchanger activity and resultant increases in intracellular [Na ϩ ] and/or pH i may mediate inotropic responses to neurohormonal stimuli, such as endothelin 1 (4), angiotensin II (5), and ␣ 1 -adrenoreceptor (␣ 1 -AR) agonists (6). Increased sarcolemmal NHE activity, as a result of the release and autocrine/paracrine actions of angiotensin II and endothelin 1 (7) or by an independent mechanism (8), may also underlie the slow increase in force development that occurs in response to myocardial stretch.With respect to cardiac pathophysiology, there is strong evidence that sarcolemmal NHE activity contributes to myocardial injury and dysfunction during ischemia and reperfusion. Thus, NHE1-selective pharmacological inhibitors, such as cariporide, have been shown to be cardioprotective in this setting in numerous animal studies (see revi...

Protein Kinase B/Akt Phosphorylates and Inhibits the Cardiac Na +/ H + Exchanger NHE1

“…Key techniques were adapted from previously published studies, including the isolation, culture and adenoviral infection of adult rat ventricular myocytes (ARVMs), 34 generation of adenoviral vectors, 35 in vitro phosphorylation assays and Western immunoblot analysis, 36 measurement of sarcolemmal NHE activity, 13 determination of cellular NHE1 phosphorylation, 36 and immunocytochemistry and confocal microscopy. 36 …”

ERK1/2-p90RSK-mediated Phosphorylation of Na+/H+ Exchanger Isoform 1

“…Analysis of NHE1 Phosphorylation by ImmunoprecipitationIn parallel, the phosphorylation level of NHE1 was determined using a method established by Snabaitis et al (18). Cells were washed with ice-cold antiphosphatase buffer and lysed in icecold immunoprecipitation lysis buffer (pH 7.5) containing (mM): 20 Tris-HCl, 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 ␤-glycerophosphate, 1 Na 3 VO 4 , 100 NaF, 1% Triton X-100, 0.1% SDS and protease inhibitors as described previously (4).…”

Harnessing the Electric Spark of Life to Cure Skin Wounds

“…60 Moreover, NHE1 is phosphorylated by several kinases which are activated by EFs, and a number of reports demonstrate that NHE1 is also a target of several phosphatases. 61,62 Interestingly, phosphorylation of NHE1 by these kinases is correlated with an increase in NHE1 activity, whereas dephosphorylation of NHE1 by protein phosphatase 1 (PP1) down-regulates NHE1 activity. 62 It is, therefore, interesting to suggest that by modulating the bal-ance between kinase and phosphatase activity, one could, in turn, modify the transmission of the electric signal and, therefore, cell behavior during ES-wound closure.…”